Abstract
Purpose :
Subretinal neovascularization (SRN) can occur in neovascular age-related macular degeneration (nAMD). The limitations of existing anti-vascular endothelial growth factor biologics for nAMD demand identification of new therapeutic targets. One promising target is apurinic/apyrimidinic endonuclease 1/reduction-oxidation factor 1 (APE1/Ref-1), the redox function of which regulates key features of nAMD, such as inflammation and angiogenesis, via transcriptionally activating HIF-1α, NF-κB, STAT3, and others. Ref-1 inhibitor APX3330 has been approved for a Phase III clinical trial for diabetic retinopathy and diabetic macular edema. We showed overexpression of Ref-1 in human nAMD and association with vasculature in mouse models of choroidal neovascularization and SRN. Here, we aimed to assess second-generation Ref-1 inhibitor APX2009 for blocking angiogenesis and whether hypoxia signaling was affected, as measured by hypoxic marker and Ref-1 responsive gene, carbonic anhydrase IX (CA9), in human retinal endothelial cells (HRECs) and in the murine Vldlr-/- SRN model.
Methods :
HRECs were treated with APX2009 (10 µM) or vehicle ± hypoxia (1% oxygen for 24 h) and mRNA (by qPCR) and protein (by immunoblot) expression of CA9 were assessed. APX2009 (12.5 and 25 mg/kg) or vehicle was injected i.p., b.i.d. in Vldlr-/- mice on postnatal days 15-21. On day 22, fundus imaging, optical coherence tomography, and fluorescein angiography (FA) were performed to assess neovascularization. Ex vivo, isolectin B4 (vasculature marker) and CA9 staining were done.
Results :
APX2009 decreased the expression of CA9 mRNA under both normoxic and hypoxic conditions. Protein levels were similarly decreased under normoxic conditions and to a lesser extent under hypoxia. Further, APX2009 reduced the number of FA-detectable SRN lesions in Vldlr-/- mice (p<0.05, n=10-12 eyes, Kruskal-Wallis test). Ex vivo, confocal image analysis showed that mean fluorescence intensity of CA9 was lowered by APX2009 (p<0.05, n=32-48 lesions, One-way ANOVA with Dunnett’s post hoc test).
Conclusions :
Ref-1 inhibition markedly downregulated expression of CA9 in HRECs under hypoxia and APX2009 decreased neovascularization and CA9 expression in Vldlr-/- mouse eyes, suggesting regulation of hypoxia-driven angiogenesis in SRN. Together, this study validates APX2009 as a potent candidate for further exploration in nAMD treatment.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.